Unite said it will affiliate 500,000 members to the party in 2014 and will review the number annually.
The union had affiliated one million members, worth £3m a year to Labour, so the move will effectively cut funding by half.
The GMB union decided last year to cut its affiliation funding by £1m because of reforms proposed by Labour leader Ed Miliband.
A special conference agreed at the weekend to adopt the changes, under which union members will have to opt in to party membership rather than be automatically affiliated.
The reforms were put forward by Mr Miliband following controversy last year over Unite's involvement in the selection of a Labour candidate in Falkirk. A Unite statement said the decision of the special conference sets the collective relationship of Unite and other unions with the party on a "new course".
Unite's executive also authorised general secretary Len McCluskey to respond to any requests for additional financial assistance from Labour, with a general election just over a year away.
The statement said: "The union will prepare a plan to ensure we maximise the number of our political levy-paying members who express support for our continuing collective affiliation, and who take advantage of becoming associated members of the party."
A Labour Party spokesman said: "We have always acknowledged that the ambitious reforms to change the Labour Party would have financial consequences.
"But we want Labour to become a party for millions, not just for millionaires."